Gene expression profiling of individual hypothalamic nuclei from single animals using laser capture microdissection and microarrays.
In order to identify novel genes involved in appetite and body weight regulation we have developed a microarray based method suitable for detecting small changes in gene expression in discrete groups of hypothalamic neurons. The method is based on a combination of stereological sampling, laser capture microdissection (LCM), PCR based amplification (SuperAmp), and one-color cDNA microarray analysis. To validate the method we assessed and compared fasting induced changes in mRNA levels of Neuropeptide Y (NPY) and proopiomelanocortin (POMC) in the hypothalamic arcuate nucleus (ARC) of diet-induced obese rats using cDNA microarrays, quantitative PCR and in situ hybridization. All methods revealed statistically significant fasting-induced changes in NPY and POMC expression. An additional 3480 differentially expressed probes (fold change >1.22, t-test p=0.05) were identified in the microarray analysis. Our findings demonstrate a consistent gene expression pattern across three different gene expression detection methods and strongly suggest that LCM coupled microarray analysis combined with SuperAmp can be used as a semi-quantitative mRNA profiling tool. Importantly, the sensitivity of the method greatly improves the usefulness of the microarray technology for gene expression profiling in non-homogeneous tissues such as the brain.
Paulsen
Sarah Juel
SJ
Rheoscience A/S, Glerupvej 1, DK-2610 Rødovre, Denmark; University of Southern Denmark, BMB, Campusvej 55, DK-5230 Odense M, Denmark. sjp@rheoscience.com
Larsen
Leif Kongskov
LK
Jelsing
Jacob
J
Janssen
Uwe
U
Gerstmayer
Bernhard
B
Vrang
Niels
N
D000704:Analysis of Variance; D000818:Animals; D001111:Arcuate Nucleus of Hypothalamus / Q000378:metabolism*; D004041:Dietary Fats / Q000009:adverse effects; D005215:Fasting / Q000502:physiology; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000502:physiology*; D008297:Male; D046228:Microarray Analysis / Q000379:methods*; D042282:Microdissection / Q000379:methods*; D009478:Neuropeptide Y / Q000235:genetics / Q000378:metabolism; D009765:Obesity / Q000209:etiology / Q000378:metabolism / Q000473:pathology; D011333:Pro-Opiomelanocortin / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014158:Transcription, Genetic / Q000502:physiology*
D004041:Dietary Fats; D009478:Neuropeptide Y; D012333:RNA, Messenger; D011333:Pro-Opiomelanocortin